Autophagy and the Degradation of Misfolded HLA-B27 Heavy Chains The propensity of HLA-B27 to misfold can generate cellular stress when sufficient heavy chains accumulate in the endoplasmic reticulum (ER). Cells often eliminate misfolded proteins through quality control pathways such as ER-associated degradation (ERAD) and autophagy. However, ERAD of HLA-B27 is insufficient to prevent heavy chains from accumulating and generating ER stress. In this study we found that HLA-B27 heavy chains expressed in HLA-B27 transgenic rats are inefficiently ubiquitinated. Since ubiquitination is necessary for ERAD, this may contribute to inadequate ER quality control of misfolded HLA-B27. We then demonstrated that autophagy is also involved in HLA-B27 degradation. Furthermore, augmentation of this pathway with rapamycin, a pharmacologic inhibitor of mTOR, promptly increases the degradation of misfolded HLA-B27. These results suggest that pharmacologic manipulation of the autophagy pathway may be exploited to alleviate the consequences of HLA-B27 misfolding, which has possible therapeutic implications. Effects of HLA-B27 on Gut Microbiota Gut microbiota have been implicated in the development of spondyloarthritis in humans and in experimental spondyloarthritis in rats. Given the major role of HLA-B27 in predisposition to disease, it has been hypothesized that this effect might be mediated by changes in gut microbiota. We asked whether HLA-B27 expressed in different strains of rats might reveal a common pattern of dysbiosis and reveal microbes that can trigger disease. We found quite unexpectedly that the different rat strains housed in different locations exhibited very different patterns of gut dysbiosis. In particular, in Fischer and Lewis rats that develop robust disease phenotypes, microbial differences associated with gut inflammation were largely non-overlapping. Despite differences in altered microbiota, informatics analysis predicted similar metabolic effects. Thus, genetic and/or environmental factors play a large role in determining effects of HLA-B27, and effects of this allele are best explained by an ecological model of dysbiosis rather than changes in one or a few organisms. Genetic Contributions to Early Onset Severe Juvenile Arthritis Whole exome sequencing revealed a de novo MYD88 germline mutation (c.666T>G, p.Ser222Arg) in a child with severe destructive polyarticular juvenile arthritis and frequent rash. MyD88 is a critical adaptor protein for TLR and IL-1 receptor signaling leading to NF-kB activation. Loss-of-function mutations in MyD88 cause severe immunodeficiency, while somatic gain-of-function mutations have been linked to certain lymphomas. We demonstrated that the mutation increases MyD88 oligomerization leading to enhanced NF-kB activation. Patient-derived cells exhibit increased STAT3 phosphorylation and overexpress chemokines and cytokines as well as interferon-responsive genes. Culture supernatants enhance neutrophil chemotactic activity. Knockdown experiments using patient cells demonstrate the dependence of the cellular phenotype on MyD88 expression, and place the biological effect distal to the Toll-IL-1 receptor accessory protein TIRAP, and proximal to the p65 subunit of NF-kB, and show that it is influenced by A20 (TNFAIP3) expression levels. Effects of the MyD88 mutation can be demonstrated in transfected cells lacking endogenous MyD88, demonstrating that it is sufficient to reproduce the cellular phenotype. In summary, we show that this germline MyD88 mutation affects hematopoietic as well as non-hematopoietic cells (fibroblasts) and is likely to contribute to the development of arthritis, although effects on the immune system are expected to be complex. These results support a role for single gene defects contributing to the pathogenesis of severe forms of juvenile arthritis. The Interaction Between HLA-B27 and ERAP1 Common loss-of-function variants of ERAP1 are associated with reduced risk of developing AS in HLA-B27 positive individuals, suggesting an important gene-gene (epistatic) interaction. To study the mechanism underlying this effect, we produced a functional ERAP1 knockout in rats using genome editing. Preliminary results suggest that ERAP1 deficiency increases presentation of longer peptides by HLA-B27, and also reduces HLA-B27 misfolding and the formation of disulfide-linked dimers and oligomers in the ER. In contrast, the folding of HLA-B7 is impaired by ERAP1 deficiency, although this does not lead to misfolding and dimerization of this allele. Preliminary results also suggest that ERAP1 deficiency confers partial protection from experimental spondyloarthritis in HLA-B27 transgenic rats, mitigating the development of arthritis but not GI inflammation, thus providing a model system to explore effects of ERAP1 loss-of-function of disease. Current studies are focused on determining whether the protective effect of ERAP1 loss-of-function on HLA-B27 folding reduces ER stress and its consequences.